Autologous fibroblasts for the treatment of cutaneous loxoscelism: First experience

Abstract

Several treatments have been described for cutaneous loxoscelism (CL), but the quality of available evidence is insufficient for the elaboration of a therapeutic consensus. Dapsone has shown beneficial effects on stopping the inflammatory phenomenon and accelerating the recovery. It is recommended to start dapsone once the visceral involvement is ruled out if glucose‐6‐phosphate dehydrogenase levels are normal. Autologous fibroblast (AF) therapy is a novel therapy that has been successfully used in bioengineering as skin substitutes for surgical wounds and burns, diabetic and pressure ulcers, and other aesthetic purposes. Interestingly, there are no reports of AFs in CL. We present a case of CL in which the necrotic process was stabilised with dapsone, but the healing of the ulcer was not achieved. Three weekly applications of AFs 100 000/cm² were performed on a biocompatible polymer matrix, with optimal response within 2 months. This represents the first report of AFs in CL, setting the stage for future studies.

1. INTRODUCTION

Evidence in cutaneous loxoscelism (CL) is scarce and of low methodological quality,1, 2 being insufficient for the elaboration of a therapeutic consensus. The venom of the Loxosceles spider contains proteins with cutaneous‐necrotising, haemolytic, vasculitic, and coagulant effects.3, 4 CL evolves with pain, oedema, and erythema within the first 6 to 8 hours in the bitten area,1 constituting afterwards the livedoid plaque, which progresses to necrosis.3 Between 4 and 6 days an eschar is formed, whose crust is released giving rise to an ulcer. The ulcer of CL is difficult to treat, usually taking more than 40 days to heal and even up to 360 days.5

Several treatments have been described for the management of CL, including corticosteroids, dapsone, colchicine, hyperbaric oxygen, antimicrobials, surgery, antihistamines, and anti‐loxosceles serum.1, 2 Dapsone is an antibiotic that belongs to the sulfone family and has bacteriostatic, anti‐inflammatory, and immunomodulatory effects.6, 7 It has been described that with dapsone lesions circumscribe faster with less pain, inflammation, necrosis, and accelerated healing of the wound.8 If, despite the abovementioned interventions, the ulcer does not heal, plastic surgery with partial or total skin grafts is usually required. Fibroblasts are critical cells during the proliferative and remodelling phases of wound healing.9 Autologous fibroblast (AF) therapy is a novel intervention that uses fibroblasts of an individual, which are extracted, cultivated, and expanded outside the body and finally reintroduced into the donor.10 Its advantages include the minimisation of immunological, infectious, and bioincompatibility risks.10 This therapy has been successfully used in bioengineering as skin substitutes for surgical wounds, acute and chronic burns, diabetic and pressure ulcers, facial wrinkles, and hair growth in androgenetic alopecia.9, 11 It is approved by the US Food and Drug Administration (FDA) to improve the appearance of nasolabial folds in adults, while other uses are still in pre‐clinical or approval stages.9 Interestingly, there are no reports of their use in CL.

2. CASE REPORT

A healthy 20‐year‐old female consulted with an eschar of CL in her left thigh, where she was bitten 13 days ago. She had been hospitalised for 1 week and treated with prednisone (40 mg/day for 5 days), amoxicillin/sulbactam, clindamycin, and chlorphenamine. It was decided to start dapsone 100 mg/day, stabilising the necrotic process in 4 days. At day 50, the ulcer was of 10 × 5 cm in diameter, with localised necrotic slough and erythematous halo, but without signs of healing. Surgical cleaning was performed with debridement of the edges and necrotic tissue, leaving a clean ulcer of 12 × 7 cm (Figure 1). Three weekly applications of AFs of 100 000/cm² were performed on a biocompatible porous polymer matrix made of gelatin, chitosan, and hyaluronic acid (Figure 2). The AFs were obtained from the patient's retroauricular area by a 3‐mm punch biopsy, the sample is immersed in a solution of Dulbecco's modified Eagle medium, and then it is transferred at 2 to 8°C to the laboratory, where they are washed with gentamicin and amphotericin B, enzymatically processed, and are seeded in a conventional cell culture for 21 days. When the desired amount of cells is obtained, they are transferred into autologous plasma within the matrix and the sterilised with gamma radiation. The Dapsone control laboratory tests were normal, so it remained at that dose. The patient evolved favourably, achieving almost complete healing in 2 months (Figure 3).

Figure 1.

Before the application of AFs

Figure 2.

First application of AFs on the biocompatible matrix

Figure 3.

Almost complete healing 4 weeks after the first application of AFs

3. DISCUSSION

According to the available evidence, the use of dapsone would prevent the accelerated progression of cutaneous necrosis and seems to be more effective than chlorphenamine or early surgery in daily doses of 50 to 200 for 10 to 25 days in adults.1 Dapsone should be initiated once visceral involvement of loxoscelism has been ruled out, mainly due to the difficulty of differentiating the origin of haemolytic anaemia, and in patients with normal levels of the enzyme glucose‐6‐phosphate dehydrogenase.2 Our patient achieved the stabilisation of the necrotic process with dapsone; however, the healing of the ulcer did not have a favourable progression.

Although there are no reports on the efficacy of AFs in CL, we decided to try due to its potential capacity to actively build the skin's architecture and its demonstrated effectiveness in acute wounds, burns, pressure, and diabetic ulcers as previously mentioned.11, 12 Our results were encouraging because the healing process was constant, fast, and the quality of the formed tissue was satisfactory. This is due to the transcendental role of fibroblasts in the wound healing process, having the ability to produce extracellular matrix proteins, differentiate into myofibroblasts to generate wound contracture, and finally synthesise type I collagen. It is not surprising that compared with allogeneic and acellular skin substitutes as treatment for full‐thickness skin defects, AFs show notably better healing and less inflammation.9, 10 A significant disadvantage of AFs is that they are not immediately available because they must be extracted and cultivated.10 Therefore, until the final coverage of the wound AF can be made, biosynthetic compounds using both autologous cells and synthetic or allogeneic materials can be used.10 A correlation has been described between the number of fibroblasts sown and the acceleration in the healing of full thickness wounds.9, 11 Excellent aesthetic and functional responses have been demonstrated with subcutaneous injection of AF 10 000 to 20 000/cm², both in acute burns and in burn scars.11, 13 Although subcutaneous injection is the most frequent way to administer AFs, its direct application in suspension in a matrix is also reported. This technique was used in our patient, as described for chronic wounds, such as diabetic and pressure ulcers. In diabetic foot ulcers, a patented mixture of cultured keratinocytes and fibroblasts is suspended in fibrin glue and applied to the wound before the area is covered with a bandage, achieving less healing time without associated side effects.9

In conclusion, evidence is scarce and of low methodological quality to guide the management of CL, but the available evidence supports the use of dapsone as a reasonable therapy. In addition, this case corresponds to the first report of the use of AFs in CL, positioning them as a future therapeutic alternative because they offer a biocompatible, safe, and simple option for tissue regeneration. Many of the possible indications of AFs are currently under investigation, and it is believed that this experience sets the stage for future studies of AFs in CL.

Guglielmetti A, Jahr C, Gompertz‐Mattar M. Autologous fibroblasts for the treatment of cutaneous loxoscelism: First experience. Int Wound J. 2019;16:1503–1505. 10.1111/iwj.13222